Coxiella burnetii, the etiologic agent of Q fever, is an obligate intraceilular bacteria that replicates within an apparently unmodified phagolysosome. Among intracellular pathogens the organisms are novel in their location of replication, extreme stability to stress, and persistence in the environment. Bacterial replication is controlled primarily by activated macrophagelmonocyte and PMN killing mechanisms stimulated by a cell mediated response, but the exact nature of these mechanisms are undefined. We propose that survival mechanisms are the principle virulence determinants of C. bumetii. We will evaluate four strategies for their contribution in survival. First, we will define the nature and role in survival of a C. bumetii lifecycle. The working hypothesis is that separable, morphological variants represent stages of cell differentiation with specific roles in intracellular and extracellular survival. Based upon earlier studies and data presented in Preliminary Studies, two major variant forms (large cell variants and small cell variants) differentially express proteins that support a model of metabolically most active dividing cells and stationary forms, respectively. Second, we will characterize the requirement for and acquisition systems used to obtain and regulate iron. The working hypothesis is that C. burnetii must accommodate conditions of limiting and high iron levels to survive in the phagolysosome. Data presented in Preliminary Studies demonstrate a C. burnetii ferric uptake regulator (fur) gene and proteins involved in iron acquisition using a ferric uptake regulator titration assay (FURTA). Third, we will characterize the role of anti-oxidant gene products in survival. The working hypothesis is that C. bumetii express enzymes which detoxify oxygen radicals outside of their cytoplasm and respond to oxidative stress by repairing DNA damage caused by oxygen radicals. Data presented in Preliminary Studies allow us to characterize catalase and RecA. We will test the molecular Koch's postulate for the requirement of RecA by creating a transdominant negative mutant through genetic transformation. Finally, we will characterize the role of acid phosphatase in intracellular survival. All specific aims will be facilitated by an ongoing genome sequence project. The working hypothesis is that C. bumetii express acid phosphatase which phosphorylates phagolysosomal proteins and reduces their antibacterial activity. It is our expectation t PERFORMANCESITE(S) (organization, city,state) Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX 77843-1114 KEY PERSONNEL. See instructions. Use conb'nua_onpages as neededto provide the requiredinformationinthe formatshownbelow. Start withPrincipal Investigator. List allotherkey personnelinalphabeticalorder,last namefirst. Name Organization Roleon Project JAMES E, SAMUEL DEPT. MED. MICRO., TAMUSHSC PI LAURA HENDRIX SAME Co-, Inv G. Q ZHANG SAME POST-DOC ROBERT BRENNAN SAME GRAD. STUDENT HEATHER BRIGGS SAME GRAD. STUDENT KERRIE SMITH SAME TECHNICIAN ANDREAS BAUMLER SAME COLLABORATOR ROBERT HEINZEN U. WYOMING-RML, NIAID COLLABORATOR Disclosure PermissionStatement. Applicable to SBIR/STTR Only. See instructions.[] Yes [] No PHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal Investigator/Program Director (Last, flint, middle): Samue I, James m_ The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,